Amplite® Luminometric Alkaline Phosphatase Assay Kit *Luminescence*

Protocol summary

1. Prepare Alkaline Phosphatase working solution (50 µL)
2. Add Alkaline Phosphatase standards and/or test samples (50 µL)
3. Incubate at RT for 30 - 60 minutes
4. Add Assay Buffer (Component D) (50 µL)
5. Incubate at RT for 10 - 30 minutes
6. Monitor luminescence intensity

Important notes
Thaw all the kit components at room temperature before starting the experiment.

1. Alkaline Phosphatase standard solution (100 U/mL):
Add 100 µL of distilled H₂O with 0.1% BSA (H₂O - 0.1% BSA) into the vial of Alkaline Phosphatase Standard (Component C, 10 units) to generate a 100 units/mL Alkaline Phosphatase standard solution. Note: The Alkaline Phosphatase standard solution is not stable.

Mix the whole content of Phosphatase Substrate (Component A) with Reaction Buffer (Component B) to make Alkaline Phosphatase working solution. Protect from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Table 1. Layout of Alkaline Phosphatase standards and test samples in a solid white 96-well microplate. AS=Alkaline Phosphatase Standards (AS1 - AS7, 0.01 to 10 mU/mL); BL=Blank Control; TS=Test Samples. 

Table 2. Reagent composition for each well.

Run alkaline phosphatase assay in supernatants:

1. Prepare Alkaline Phosphatase standards (AS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
   
   
2. Add 50 µL of Alkaline Phosphatase working solution to each well of Alkaline Phosphatase standard, blank control, and test samples to make the total Alkaline Phosphatase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Alkaline Phosphatase working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction for 30 to 60 minutes at room temperature, protected from light.
   
   
4. Add 50 µL of Assay Buffer (Component D) into each well of Alkaline Phosphatase standard, blank control, and test samples with assay reaction mixture to make the total Alkaline Phosphatase assay volume of 150 µL/well. For a 384-well plate, add 25 µL of Assay Buffer (Component D) into each well, for a total volume of 75 µL/well.
   
   
5. Incubate the reaction for 10 to 30 minutes at room temperature, protected from light.
   
   
6. Monitor the luminescence increase with a standard luminescence plate reader.

Run alkaline phosphatase assay in cells:

1. Treat the cells as desired.
   
   
2. Remove the growth medium completely from the cell plate. Note: It is important to remove the growth medium completely from the cell plate due to the interference of the growth medium with the phosphatase substrate.
   
   
3. Make 1:1 dilution of the 5 mL Alkaline Phosphatase working solution with 5 mL distilled H₂O.
   
   
4. Add 100 µL (96-well plate) or 50 uL (384-well plate) of 1:1 diluted Alkaline Phosphatase working solution into the cell wells.
   
   
5. Incubate the reaction at the desired temperature for 30 to 60 minutes, protected from light.
   
   
6. Add 50 µL (96-well plate) or 25 uL (384-well plate) of Assay Buffer (Component D) into the cell wells containing working solution.
   
   
7. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
   
   
8. Monitor the luminescence increase with a standard luminescence plate reader.